Advances in recombinant DNA technology and genetic engineering have provided a means for producing in bacteria eucaryotic proteins of clinical and hence economic importance. The employment of bacterial cells as factories (e.g. host cells) for eucaryotic protein production has become especially attractive for eucaryotic proteins of limited availability. One important example of limited availability is human hormones. The problem of obtaining tissue is further magnified by the fact that a given tissue extract yields very low quantities of a given hormone.
The use of bacteria as host cells for eucaryotic protein production currently involves first isolating or synthesizing the gene or DNA sequence encoding the desired peptide and, second, incorporating the gene or DNA sequence into the genome of the host cell in a manner which allows for expression of the DNA sequence or gene and resultant protein production, accumulation and/or secretion.
Differences between eucaryotic and procaryotic cellular control of gene expression and protein production, however, have given rise to several obstacles which must be overcome if given eucaryotic proteins, peptides or fragments thereof are to be produced in bacteria efficiently and at commercially-attractive levels.
In eucaryotes, many mature proteins are first translated as pre-proteins; i.e., polypeptides comprised of the mature proteins's sequence fused to a leader or signal sequence. Eucaryotic mRNA encodes the entire pre-protein, which is processed after translation to remove the leader sequence and provide the mature protein. Although eucaryotic cells are equipped to specifically process such pre-proteins into mature proteins, bacterial cells are generally not able to recognize the processing signals present in eucaryotic proteins. Thus, if complete complementary DNA (cDNA) transcripts of eucaryotic mRNA are employed as the DNA sequences for expression in bacteria, the pre-protein, not the mature protein, is obtained. It is possible to convert pre-proteins to mature proteins in vitro, but not without significant expense.
In the event that the DNA sequence encoding the mature protein is used for mature protein expression in bacteria, this sequence will be lacking the eucaryotic translation and post-translation processing signals usually contained within the DNA for the leader sequence. Therefore, for expression of cloned eucaryotic genes or other heterologous DNA sequences in bacterial systems, it has proven desirable to employ bacterial control signals for reasons of efficiency and because eucaryotic signals may not be recognized by a bacterial host cell.
The term "heterologous DNA" is defined herein as DNA at least a portion of which is not normally contained within the genome of the host cell. Examples of heterologous DNA include, but are not limited to, viral and eucaryotic genes, gene fragments, alleles and synthetic DNA sequences. The term "heterologous protein" or "heterologous polypeptide" is defined herein as a protein or polypeptide at least a portion of which is not normally encoded within the genome of the host cell. The term "genome" refers to all DNA (chromosomal and extrachromosomal) contained within a specified cell.
The bacterial control signals include a promoter, which signals the initiation of transcription, and translation control signals comprising a ribosome binding site, a translation start signal and a translation stop signal. All of these signals except the translation stop signal must be situated in front of the eucaryotic gene or other DNA to be expressed.
The art has adopted several approaches to expressing heterologous DNA (e.g. eucaryotic genes) in bacteria. In one approach, the translation start signal, ATG, under the control of a bacterial promoter, is located immediately preceding the DNA sequence encoding a heterologous (e.g. eucaryotic) protein. Expression of such a DNA construct results in production of eucaryotic proteins free from endogenous proteins or protein fragments herein defined as "direct" protein production. The proteins so produced, however, typically contain an amino(N)-terminal methionine as the ATG translation start signal is also a methionine codon. See Harris, T.J.R. (1983). Thus, unless the desired mature protein begins with methionine, the protein will now have an N-terminus altered by inclusion of that methionine residue.
Additionally, the direct production approach has not generally been successfully applied to production of heterologous (e.g. eucaryotic) peptides, wherein a "peptide" is defined as a protein containing fewer than 100 amino acids or proteins having a molecular weight of less than about 10,000 daltons. The problem with direct production of heterologous peptides lies in the tendency of bacteria, such as E. coli, to recognize eucaryotic peptides produced therein as foreign and, thus, proceed to degrade these peptides as soon as these peptides are produced or shortly thereafter. See. R.K. Craig and L. Hall (1983); Itakura et al. (1977). Furthermore, it has been found that structural features inherent in the nucleic acid (DNA or RNA) sequence coding for a heterologous protein or peptide product often interfere with efficient heterologous protein or peptide production (i.e. translation) in bacteria. Hence, an alternate approach to production of such heterologous peptides as eucaryotic peptide hormones has been developed.
In one alternate approach, the DNA segment encoding the desired protein or peptide is ligated to endogenous DNA encoding all or part of a bacterial protein under the control of its bacterial promoter. The endogenous bacterial DNA necessarily also contains the ribosome binding site and translation start signal. In ligation, the DNA encoding the desired protein or peptide must be inserted in-frame with the endogenous transcription and translation control signals and endogenous DNA coding sequences, and in the same orientation. Expression of the ligated DNA provides a fusion protein comprising the heterologous protein or peptide linked (e.g. fused) to a whole or partial bacterial protein. Ideally, such fusion constructs should provide a relatively high and/or stable level of fusion protein accumulation in the bacterial host cell and/or high level of secretion by the host cell.
Production of heterologous proteins and peptides in bacteria has been reported to be aided by fusion of the desired heterologous product peptide or protein to an endogenous protein or fragment thereof. For example, the endogenous protein may serve to enhance transcription and/or translation, Craig and Hall (1983), or, may be employed to aid in purification of desired product. See Sessenfeld, H. M. and Brewer, S. J. (1984) (use of polyarginine binding to ion exchange columns); Germino, J. and Bastia, D. (1984) (.beta.-galactosidase affinity column). In addition, especially in Bacillus and yeast systems, fusion of a desired protein or peptide to an endogenously secreted protein or a signal peptide may result in the secretion of mature protein product into the host growth media free from intracellular proteins and endogenous protein sequences.
Furthermore, the fusion protein approach is useful in protecting otherwise foreign protein or peptide products from intracellular degradation. See Itakura, K. et al. (1977) and R. K. Craig and L. Hall (1983). Fusion proteins engineered for protective purposes can employ endogenous polypeptide sequences at either the amino or carboxy terminus of the heterologous peptide.
In all cases, final isolation of the bacterially-produced eucaryotic peptide must be achieved by site-specific enzymatic or chemical cleavage at the endogenous-eucaryotic peptide fusion site, herein referred to as "junction site", or by selective degradation of the endogenous polypeptide sequences. The junction site may contain a single peptide bond that links the heterologous (e.g. eucaryotic) peptide to the endogenous protein or contain a series of peptide bonds joining the heterologous peptide to the endogenous protein. Most commonly, bacterially produced fusion proteins are constructed so that the endogenous peptide or fragment thereof comprises the N-terminal portion of the fusion protein with the heterologous peptide comprising the C-terminal portion. Such constructions allow for the simultaneous release of the endogenous peptide/protein and the N-terminal methionine following cleavage at the junction site.
Examples of site-specific release of eucaryotic peptides from bacterially produced fusion proteins by chemical means include the following: Stephien et al. (1983) (proinsulin fused to yeast galactokinase); Tanaka et al. (1982) (.alpha.-neo-endorphin fused to E. coli .beta.-galactosidase); Goeddel et al. (1979) (insulin A and B chain fused to E. coli .beta.-galactosidase); Itakura et al. (1977) (somatostatin fused to E. coli .beta.-galactosidase). In all the foregoing examples, the chemical cyanogen bromide was employed to cleave the fusion protein and release the desired peptide. Cleavage of a protein or polypeptide is defined herein as the hydrolysis of a peptide bond in a protein or polypeptide. Cyanogen bromide hydrolyzes peptide bonds at the carboxy-side of methionine residues under acid conditions. Thus, site-specific cleavage of a fusion protein requires the presence of a methionine residue immediately upstream and adjacent to the N-terminal amino acid of the desired peptide and an absence of methionine residues in the internal amino acid sequence of the desired peptide.
The disadvantages of chemical hydrolysis include the harsh acid conditions under which cleavage occurs, such conditions possible causing undesirable modifications in the product peptide, the need to know the amino acid sequence of the product peptide to insure against internal cleavage sites, and the observation that the specificity of some chemical cleavages depend largely upon amino acids immediately adjacent to the bond being cleaved.
As an alternative to chemical cleavage, several investigators have reported the use of enzymes, peptidases, to achieve release of the desired peptide product from bacterially produced fusion proteins. Peptidases are generally defined as enzymes which catalyze the hydrolysis (cleavage) of peptide bonds.
One specific class of peptidases employed to date has been the endopeptidases. These peptidases are particularly well suited for use in release of a desired peptide from fusion proteins comprising an endogenous (carrier) protein at the N-terminus of the fusion protein and the desired peptide at the C-terminus. Endopeptidases recognize either specific single amino acids or specific amino acid sequences present within the internal amino acid sequence of a polypeptide and then cleave the peptide bond preferably on the carboxyside of a given amino acid. The amino acid or amino acid sequence specifically recognized and cleaved by a given endopeptidase shall henceforth be referred to as a "trigger amino acid" or "trigger sequence", respectively, or collectively as a "trigger signal."
Examples of various endopeptidases employed to cleave bacterially produced fusion proteins to release a desired peptide include the following: International Patent Application publication number WO84/00380 (published Feb. 9, 1981) (trypsin to release human calcitonin from a tryptophan promoter/operator system); European Patent Application publication number 35,384 (published Sept. 9, 1981) (suggested use of enterokinase); Nagai and Thogersen (1984) (Factor Xa to release human .beta.-globin from a .lambda.CII protein); Germino, J. and Bastia, D. (1984) (microbial collagenase to release R6K replication initiator from .beta.-galactosidase); Shine et al. (1980) (trypsin to release .beta.-endorphin from .beta.-galactosidases); Rutter, W. J. (1979) (suggested use of enterokinase to cleave fusion proteins); European Patent Application publication number 161,937 (published Nov. 21, 1985) (Factor Xa to release .beta.-globin from .lambda.CII, human calcitonin glycine from CAT, and myosin light chain from .lambda.CII).
As in the case of chemical cleavage, the trigger signal must constitute the junction bond or site if release of the mature peptide from the bacterially produced fusion protein is to be achieved. Unlike chemical cleavage, however, the vast number of endopeptidases available affords a greater choice of trigger signals for potential use in peptide release.
The decision of which trigger signal or endopeptidase is best employed to achieve release is governed by several factors most of which are tied to the specific bacterial expression system employed to produce the fusion protein and the amino acid sequence of the desired peptide itself. As will be discussed herein, there exists a significant degree of unpredictability in the art. This predictability is best understood by reviewing some of the factors which affect selection of a given endopeptidase and subsequent cleavage of fusion proteins by endopeptidases.
The major factors affecting the choice of the trigger signal and hence endopeptidase employed is whether the complete amino acid sequence of the desired peptide is known and whether the resultant fusion protein allows endopeptidase cleavage at the junction site. If the amino acid sequence is known, a trigger signal can be chosen which does not occur in the desired peptide thereby avoiding unwanted hydrolysis of the desired peptide. Once chosen, a DNA sequence encoding the trigger signal must be synthesized and inserted at the junction site in a manner which will not significantly interfere with expression, production, accumulation and/or purification of the fusion protein. For example, when inserting a given trigger signal at the junction site, the insertion must not disturb the in-frame reading of the coding sequences for the endogenous and heterologous peptides. In preparing the fusion protein for endopeptidase cleavage, it is necessary that the trigger signal be available or exposed for optimum cleavage and release of the desired peptide, and that the cleavage conditions be such that the desired peptide is not irreparably damaged by the reaction conditions necessary for cleavage. Additionally, it is generally desirable that trigger signals are not present within the endogenous protein so that a clean release of the desired peptide can be achieved. The maintenance of the integrity of the endogenous protein is often required for a subsequent commercially feasible purification of the desired peptide.
In the event that the precise amino acid sequence of the desired peptide is not known, the trigger signal should comprise an amino acid sequence of sufficient complexity so as to diminish the likelihood of a similar sequence being contained within the desired peptide.
As an additional consideration when employing endopeptidases, it has been determined that for some endopeptidases, amino acids in the vicinity of the site of hydrolysis will be recognized and/or bound by the enzyme. These "peripheral" amino acids, in some instances, can increase the catalytic efficiency or binding affinity of the enzyme and thus effect the susceptibility of a peptide containing a trigger signal to hydrolysis by a given endopeptidase. European Patent Application publication number 35,384 (published 9/9/81). Conversely, these peripheral amino acids may decrease the hydrolytic efficiency of a given endopeptidase. Behrens and Brown (1976); Austen and Smith (1976); Houmard and Drapeau (1972a). The presence of peripheral amino acids at or near the junction site should, therefore, be considered as to their effects, if known, on the trigger signal. The effect of peripheral amino acids on specific endopeptidase cleavage is, however, unknown for many of the described endopeptidases. Thus, the ambiguities in the context (e.g. structure) and content (e.g. linear sequence) of any given endopeptidase trigger signal render the operability of a trigger signal for release of a desired peptide from a fusion protein unpredictable in most instances.
In summary, construction of a specific fusion protein with a given trigger signal providing for site-specific release of a desired peptide must accommodate a plurality of factors affecting not only endopeptidase cleavage but polypeptide expression, production and accumulation as well. A fusion protein system applicable to the production of a wide variety of desired proteins or peptides does not currently exist which will satisfy all these factors.
As indicated earlier, one important embodiment of the claimed invention involves production of atrial peptides as a bacterially produced fusion protein, cleavage of the fusion protein and recovery of the product. Mammalian atria contain peptides that exert potent effects on kidney function and regional vascular resistance. These peptides, originally extracted from rat atria and exerting natriuretic, diuretic and smooth muscle relaxant (e.g. vasodilating) activities are currently referred to as atrial peptides.
Rat atrial extracts have been fractionated into low molecular weight fractions (&lt;10,000 daltons) and high molecular weight fractions (20,000-30,000 daltons) both of which relaxed smooth muscle in vitro and were potent natriuretic agents when administered intravenously to rats. See Currie et al. (1983). Trippodo et al. (1982) found natriuretic activity in the overall molecular weight range of 3,600 to 44,000 daltons and in peptide fractions of both higher molecular weight range of 36,000-44,000 daltons and a lower molecular weight in the range of 3,600-5,500 daltons.
Efforts devoted to the purification and chemical characterization of atrial peptides have been hampered by the scarcity of material available from atrial homogenates and by the apparent heterogeneity of the biologically active factor. The amino acid sequence of several atrial peptides is now known, see U.S. Pat. No. 4,496,544; U.S. Pat. No. 4,508,712; European Patent Application publication number 116,784 (published 8/29/84); Seidah, N.G. et al. (1984); deBold et al. (1983); deBold and Flynn (1983). The low molecular weight nature (&lt;10,000 daltons) of many of the atrial peptides identified to date will undoubtedly require the fusion protein approach for production in bacteria.
Accordingly, it is an object of the present invention to provide endopeptidases useful in achieving optimum cleavage of a bacterially produced fusion protein to release a desired heterologous peptide.
It is another object of the present invention to provide methods for cleaving bacterially produced fusion proteins to achieve the release of desired heterologous peptides in their mature form.
It is yet another object of the invention to provide methods for producing in bacteria atrial peptides having useful natriuretic, diuretic and/or vasodilating activity.
It is a further object of the present invention to provide methods for producing in bacteria, fusion proteins containing atrial peptides which have useful natriuretic, diuretic and/or vasodilating activity.
It is still a further object of the present invention to provide methods for producing in bacteria fusion proteins containing atrial peptides wherein said atrial peptides can be enzymatically released from said fusion protein in their mature form.
It is yet a further object of the present invention to provide fusion proteins which allow for high level of production, in bacteria, of atrial peptides and affords site-specific cleavage and release of atrial peptides from the fusion protein.